Harnessing transthyretin to extend the in vivo half-life of therapeutic peptides PROJECT SUMMARY: The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmacokinetic (PK) profile, due to short in vivo half-life resulting from inactivation by serum proteases and rapid elimination by kidney. Therefore, extending the in vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and/or frequent administration. Covalent conjugation of peptides to macromolecules (e.g. polyethylene glycol or serum proteins) has been the mainstay approach for enhancing the in vivo half-life of peptides. However, the steric hindrance of these large macromolecules often harms the binding of the peptide to its extra-cellular receptor, which compromises the pharmacodynamic (PD) properties of the peptide. The overall goal of this proposal is to develop a fundamentally new approach for enhancing the in vivo half-life of peptides without affecting its biological activity. The main hypothesis of this proposal isto test whether conjugation of peptides to a selective transthyretin (TTR, a serum protein) ligand would allow these peptide conjugates to reversibly bind to TTR in serum. This will result in enhancing the peptide's in vivo half-life, but most importantly, will maintain the binding affinityof the peptide to its receptor. A major emphasis will be on our recent discovery of the most potent and selective reversible TTR ligand to date, AG10. We have already made significant progress by synthesizing the first AG10-peptide conjugate (Conj1; with GnRH as a model peptide) and our in vitro preliminary results for Conj1 are consistent with this hypothesis. In this proposal, w will evaluate Conj1 in vivo and investigate the optimal linker system and affinity to TTR that will provide best balance between PK and PD of these AG10-GnRH conjugates. Three specific aims will be aggressively pursued: aim 1, design and synthesize a series of AG10-GnRH conjugates and determine the optimal linkers length and conjugation sites (on AG10 and GnRH) that will preserve the maximum binding affinity and serum selectivity of conjugates to both TTR and GnRH receptor; aim 2, evaluate the in vitro half-life of conjugates in serum and determine the optimal linker system that would allow for best balance between protection of GnRH against serum proteases and binding affinity to TTR and GnRH receptor; and aim 3, evaluate the pharmacokinetic properties of conjugates to determine if selective binding to TTR will increase the in vivo half-life, while maintaining the efficacy of conjugates in rats. If we successfully accomplished the goal of this proposal, our approach would represent an innovative technology that could potentially be applicable to enhancing the in vivo half-life of proteins, oligonucleotids, oligosaccharides, virus like particle, imaging agents, and other small molecule drugs.